Phosphohalohydrins, process for the production thereof and use thereof

ABSTRACT

The invention provides compounds comprising at least one phosphohalohydrin group of the formula: 
                         
where X is a halogen selected from among I, Br, Cl,
         R 1  is selected from among —CH 3  and —CH 2 —CH 3 ,   Cat+ is an organic or inorganic cation,   and n is an integer between 2 and 20,
 
processes for the production thereof and uses thereof, in particular therapeutic uses and for activating primate Tγ9δ2 lymphocytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/682,990, filed Oct. 14, 2003, now U.S. Patent No. 7,109,183, which isa divisional of U.S. patent application Ser. No. 09/786,054, filed onMar. 1, 2001, now U.S. Patent No. 6,660,723, which is the national stageapplication of PCT International Application No. PCT/FR99/02058, filedon Aug. 27, 1999. The entire contents of each of the above-identifiedapplications are hereby incorporated by reference.

This invention relates to novel phosphohalohydrins, to the process forthe production thereof and to the use thereof for stimulating Tγ9δ2lymphocytes bearing TCR receptors with Vγ9 and Vδ2 variable regions.

In healthy individuals, the Tγδ lymphocytes of primates (humans,monkeys) present in the peripheral bloodstream usually constitute from 1to 5% of the lymphocytes in the blood and play a role in the immunesystem. It has been demonstrated that they recognize their antigenicligands by direct interaction with the antigen without presentation bymolecules of the MHC by a presenting cell. Tγ9δ2 lymphocytes (sometimesalso known as Tγ2δ2 lymphocytes) are Tγδ lymphocytes bearing TCRreceptors with Vγ9 and Vδ2 variable regions. They constitute themajority of Tγδ lymphocytes in human blood.

When activated, Tγδ lymphocytes exercise a strong cytotoxic activitywhich is unrestrained by the MHC and is particularly effective inkilling various types of cells, in particular pathogenic cells. Thesemay be cells infected by viruses (“γδ T cell activation or anergy duringinfections: the role of nonpeptidic TCR ligands and HLA class Imolecules” Fabrizio POCCIA et al, Journal of Leukocyte Biology, 62,1997, p. 1-5), or by other intracellular parasites, such as mycobacteria(“The antituberculous Mycobacterium bovis BCG Vaccine is an attenuatedMycobacterial producer of phosphorylated nonpeptidic Antigens for humanγδ T cells” Patricia CONSTANT et al, Infection and Immunity, vol. 63,no. 12, December 1995, p. 4628-4633); or by protozoans (“Plasmodiumfalciparum stimuli for human γδ T Cells are related to phosphorylatedAntigens of mycobacteria” Charlotte BEHR et al, Infection and Immunity,Vol. 64, no. 8, 1996, p. 2892-2896). They may also be cancer cells(“CD94/NKG2 inhibitory receptor complex modulates both antiviral andantitumoral responses of polyclonal phosphoantigen-reactive Vγ9 Vδ2 Tlymphocytes” Fabrizio POCCIA et al, Journal of Immunology, 159, p.6009-6015; “Stimulation of γδ T cells by phosphoantigens” Jean-JacquesFOURNIE, Marc BONNEVILLE, Res. Immunol., 66^(th) FORUM IN IMMUNOLOGY,147, p. 338-347).

It has been demonstrated that, in the event of a mycobacterialinfection, human Tγ9δ2 lymphocytes react to four natural, nonpeptidicmolecules of a phosphorylated structure, known as phosphoantigens, whichexhibit stimulation activity at a concentration of 1 to 5 nM (nanomolar)(WO-95/20673 and “Stimulation of human γδ T cells by nonpeptidicMycobacterial ligands” Patricia CONSTANT et al, Science, 264, p.267-270).

These natural antigens have not been completely identified. Certainauthors have erroneously presented them as alkene derivatives ofpyrophosphate, in particular isopentenyl pyrophosphate IPP (U.S. Pat.No. 5,639,653 and “Natural and Synthetic nonpeptide antigens recognizedby human γδ T cells”, Yoshimasa TANAKA et al, Nature, 375, 1995, p.155-158). It has nonetheless now been demonstrated that none of theseprenyl pyrophosphates is active at a concentration of nanomolarmagnitude. The best results which have been obtained have been unable todemonstrate activity at below 3 μM for IPP and below 0.3 μM fordimethylallyl-UTP and 3-methyl-2-hexene pyrophosphate. The minimumactive concentration of these compounds is thus, at best, of the orderto 100 times higher than that of natural phosphoantigens.

With regard to IPP, it should in particular be noted that the mostrecent of the above-stated publications make the mistake of deducing thestructure of the isopentenyl radical solely on the basis of massspectrometry and the detection of a certain level of bioactivity.Indeed, apart from the fact that the compound analyzed in thesepublications was not purified and that a mass spectrum cannot identifyuncharged species, it may be demonstrated that there are in fact severalthousand different chemical structures which may have the same molecularweight and be a substituent of pyrophosphate in these molecules.

The fact that the minimum active concentration for IPP is much higher(some 1000 times higher) and that the intensity of the Tγ9δ2 lymphocyteresponses obtained is much weaker than that for natural phosphoantigensdemonstrates that IPP is not one of these natural phosphoantigens (“Anovel nucleotide-containing antigen for human blood γδ T lymphocytes”,Y. Poquet et al, Eur. J. Immunol. 1996, 26, p. 2344-2349). This ismoreover confirmed by numerous other observations: IPP is not found insufficient concentration in mycobacterial extracts which stimulate Tγ9δ2lymphocytes; according to “High pH anion exchange chromatographicanalysis of phosphorylated compounds: application to isolation andcharacterization of non peptide mycobacterial antigens”, Y. Poquet etal, Anal. Biochem, 243 no. 1, 1996, p. 119-126, IPP does not have thesame chromatographic (HPAEC) characteristics as natural phosphoantigens;IPP and other natural isoprenoids are produced by all living cells, butthese do not stimulate Tγ9δ2 lymphocytes.

Moreover, it is known that substances having bioactivity of the order ofor greater than 1 μM are only rarely compatible with the economicconstraints of operation on an industrial scale. The syntheticphosphoantigens which have hitherto been proposed thus cannot beprocessed on an industrial scale under acceptable economic conditions.

Natural phosphoantigens, on the other hand, may only be produced in verysmall quantities (WO 95/20673) and, since their precise chemicalstructure has still not yet been established, they cannot besynthesized. Economic industrial scale processing is thus likewise outof the question, despite their demonstrated great therapeutic worth.

The object of the invention is accordingly to provide novel chemicalcompounds which activate Tγ9δ2 lymphocytes at a minimum activationconcentration of below 100 nM, in particular of the order of 1 nM.

A further object of the invention is to provide compounds which may belinked to a large number of organic groups, in particular to natural orsynthetic peptide groups, so as to permit multifunctional compounds tobe obtained.

A further object of the invention is to provide such compounds which maybe synthesized simply, quantitatively and at low cost, i.e. in a mannercompatible with the economic constraints of production on an industrialscale.

A further object of the invention is to provide an advantageoussynthetic pathway for these compounds.

A further object of the invention is to provide a process for theproduction of the compounds according to the invention.

A further object of the invention is to suggest uses for the compoundsaccording to the invention as a Tγ9δ2 lymphocyte activator and inparticular therapeutic uses of the compounds according to the invention.

The invention accordingly provides compounds comprising at least onephosphohalohydrin group of the formula:

where X is a halogen selected from among iodine, bromine and chlorine,

-   -   R1 is selected from among —CH₃ and —CH₂—CH₃,    -   Cat⁺ represents one or more organic or inorganic cation(s)        (including the proton), which may be identical or different in        the same compound,    -   and n is an integer between 2 and 20.

A compound according to the invention may in particular comprise one ormore phosphohalohydrin group(s) selected from among the esters of thefollowing groups (IUPAC nomenclature) or among the compounds formed fromthese groups:

-   3-(halomethyl)-3-butanol-1-yl diphosphate,    3-(halomethyl)-3-pentanol-1-yl diphosphate,    4-(halomethyl)-4-pentanol-1-yl diphosphate,    4-(halomethyl)-4-hexanol-1-yl diphosphate,    5-(halomethyl)-5-hexanol-1-yl diphosphate,    5-(halomethyl)-4-heptanol-1-yl diphosphate,    6-(halomethyl)-6-heptanol-1-yl diphosphate,    6-(halomethyl)-6-octanol-1-yl diphosphate,    7-(halomethyl)-7-octanol-1-yl diphosphate,    7-(halomethyl)-7-nonanol-1-yl diphosphate,    8-(halomethyl)-8-nonanol-1-yl diphosphate,    8-(halomethyl)-8-decanol-1-yl diphosphate,    9-(halomethyl)-9-decanol-1-yl diphosphate,    9-(halomethyl)-9-undecanol-1-yl diphosphate,    10-(halomethyl)-10-undecanol-1-yl diphosphate,    10-(halomethyl)-10-dodecanol-1-yl diphosphate,    11-(halomethyl)-11-dodecanol-1-yl diphosphate,    11-(halomethyl)-11-tridecanol-1-yl diphosphate,    12-(halomethyl)-12-tridecanol-1-yl diphosphate,    12-(halomethyl)-12-tetradecanol-1-yl diphosphate,    13-(halomethyl)-13-tetradecanol-1-yl-diphosphate,    13-(halomethyl)-13-pentadecanol-1-yl diphosphate,    14-(halomethyl)-14-pentadecanol-1-yl diphosphate,    14-(halomethyl)-14-hexadecanol-1-yl diphosphate,    15-(halomethyl)-15-hexadecanol-1-yl diphosphate,    15-(halomethyl)-15-heptadecanol-1-yl diphosphate,    16-(halomethyl)-16-heptadecanol-1-yl diphosphate,    16-(halomethyl)-16-octadecanol-1-yl diphosphate,    17-(halomethyl)-17-octadecanol-1-yl diphosphate,    17-(halomethyl)-17-nonadecanol-1-yl diphosphate,    18-(halomethyl)-18-nonadecanol-1-yl diphosphate,    18-(halomethyl)-18-eicosanol-1-yl diphosphate,    19-(halomethyl)-19-eicosanol-1-yl diphosphate,    19-(halomethyl)-11-heneicosanol-1-yl diphosphate,    20-(halomethyl)-20-heneicosanol-1-yl diphosphate,    20-(halomethyl)-20-docosanol-1-yl diphosphate,    21-(halomethyl)-21-docosanol-1-yl diphosphate,    21-(halomethyl)-21-tricosanol-1-yl diphosphate.

The phosphohalohydrin compounds in accordance with one of the followingformulae may be mentioned among the compounds according to theinvention:

where R2 is an organic or inorganic substituent selected from among thegroup comprising:

-   -   substituents which do not prevent formation of the halohydrin        function

starting from the alkene function

and halogen X₂ in the presence of water;

-   -   and substituents for which there is an R2—O—Y compound which is        not reactive towards the halohydrin function of the compound of        the formula:

and selected such that R2—O—Y may react with the terminal phosphate ofthis compound (3) in order to obtain the compound (4).

Said compounds according to the invention are advantageouslycharacterized in that n=2 and R1 is CH₃.

The compounds according to the invention advantageously additionallycomprise at least one group selected from among the group comprisingnucleoside derivatives, oligonucleotides, nucleic acids (RNA, DNA),amino acids, peptides, proteins, monosaccharides, oligosaccharides,polysaccharides, fatty acids, simple lipids, complex lipids, folic acid,tetrahydrofolic acid, phosphoric acids, inositol, vitamins, co-enzymes,flavonoids, aldehydes, epoxides and halohydrins.

In particular, the invention provides the phosphohalohydrin compounds ofthe formula (4) above in which R2 is selected from among the groupcomprising nucleoside derivatives, oligonucleotides, nucleic acids (RNA,DNA), amino acids, peptides, proteins, monosaccharides,oligosaccharides, polysaccharides, fatty acids, simple lipids, complexlipids, folic acid, tetrahydrofolic acid, phosphoric acids, inositol,vitamins, co-enzymes, flavonoids, phosphohalohydrins of the formula (1),aldehydes, epoxides and halohydrins.

The invention also provides compounds, the structure of whichincorporates two or more groups of the formula (1), which may beidentical or different, for example monomers, polymers, oligomers ordendrimers, or more generally molecules with two or more phosphorylatedbranches of the formula (1).

It should be noted that the compounds according to the invention areesters (monoesters or diesters) of phosphoric acid (this termencompassing those acids in which phosphorus is in oxidation state V,namely orthophosphoric acid, pyrophosphoric acid, metaphosphoric acid,triphosphoric acid, other polyphosphoric acids).

The invention provides a process for the production of the compoundsaccording to the invention. According to the invention, halogen X₂ isreacted in the presence of water with a starting compound comprising atleast one phosphorylated alkene group of the formula:

Advantageously and according to the invention, a salt formed from saidstarting compound is reacted in an aqueous or aqueous/alcoholic medium,at neutral pH, at a temperature of below 30° C., by mixing with anaqueous solution of the halogen X₂. Advantageously and according to theinvention, the reaction is performed at atmospheric temperature at atemperature of between 0° C. and 25° C.

The starting compounds may themselves be obtained from alcohol:

Advantageously and according to the invention, the starting compound isa salt of the formula:

The pyrophosphohalohydrin compound of the formula (2) is then obtained.

An example of a complete reaction scheme for obtaining the compound (2)from the alcohol (9) is given below:

-   -   where TsCl is tosyl chloride,    -   4-DMAP is 4-dimethylaminopyridine,    -   Bu₄ N+ is tetrabutylammonium,    -   (Bu₄N+)₃ HP₂O₇ is tris(tetra n-butylammonium)        hydrogenpyrophosphate,    -   PP represents the pyrophosphate group.

The reactions which allow compound (6) to be obtained from the alcohol(9) may be performed as described by: DAVISSON V. J. et. al.“Phosphorylation of Isoprenoid Alcohols” J. Org. Chem 1986, 51,4768-4779, et DAVISSON V. J. et al. “Synthesis of Allylic andHomoallylic Isoprenoid Pyrophosphates” Methods in Enzymology, 1984, 110,130-144.

Advantageously and according to the invention, the starting compound isa salt of the formula:

The triphosphohalohydrin compound of the formula (3) is then obtained.

An example of a complete reaction scheme for obtaining the compound (3)from the alcohol (9) is given below:

where PPP is the triphosphate group,

(Bu₄N⁺)₄HP₃O₁₀ is tetralcis(tetra-n-butylammonium) hydrogentriphosphate.

The compound (10) is obtained from the alcohol (9) as stated above. Thereaction which allows compound (7) to be obtained from the compound (10)may be performed under conditions similar to those described in thepublications by DAVISSON V. J. et al or in DAVISSON V. J. et al“Synthesis of Nucleotide 5′-Diphosphates from (5′-O-Tosyl Nucleosides”J. Org. Chem. 1987, 52, 1794-1801.

Advantageously and according to the invention, in a first variant whichallows a compound according to the invention of the formula (4) to beobtained, the above-stated production process according to the invention(reaction of X₂ in the presence of water with a phosphorylated alkenefunction) may be performed by using a salt of the following formula asthe starting compound:

where R2 is an organic or inorganic substituent of a nature such as notto prevent formation of the halohydrin function

starting from the alkene function

and halogen X₂ in the presence of water.

The starting compound (8) may itself be prepared in accordance with oneof the following reaction schemes:

where Ts is tosyl.

The compound (7) may be obtained as stated above starting from thealcohol (9) and the intermediate compound (10). The reaction whichallows compound (8) to be obtained from the compound (7) may beperformed under conditions similar to those described in thepublications by DAVISSON V. J. et al. This scheme may be used whenR2—O-Ts is commercially available.

The intermediate compound (10) may be obtained as stated above startingfrom the alcohol (9). The reaction which allows compound (8) to beobtained from the compound (7) may be performed under conditions similarto those described in the publications by DAVISSON V. J. et al. Thisscheme may be used when R2—O—PPP is commercially available.

where DMF is dimethylformamide,

MeOH is methanol.

This reaction scheme 3 may be performed under conditions similar tothose described in D. G. KNORRE et al “General method for the synthesisof ATP gamma derivatives” Febs letters, 1976, 70, 105-108.

This reaction scheme 3 cannot be used when R2 comprises acarbodiimide-reactive function (carboxylate, triphosphate etc.). It is,however, advantageous when R2—O—PPP is commercially available.

In the specific case where R2— is itself a halohydrin group of theformula:

the following reaction scheme may be used:

This compound (4′) is a particular instance of the compound according tothe invention of the formula (4).

It should be noted that, in all these reactions, acetonitrile may bereplaced by any other aprotic dipolar solvent (dimethylformamide DMF,dimethyl sulfoxide DMSO etc.).

It should be noted that when preparing compounds (2), (3) and (4′) andin the event that n≠2, the intermediate compound (10) may also bereplaced by the chloride or bromide compound of the formula:

-   -   where A is chlorine or bromine.

The alcohols (9) are commercially available or may readily be obtainedby a well known Grignard reaction between an alkenyl organomagnesiumcompound and formaldehyde or ethylene oxide.

In a second variant usable in certain cases, a compound of the formula(4) could be prepared by reacting the triphosphate compound according tothe invention of the formula (3) starting from a salt soluble in anorganic medium, such as a Bu₄N+ salt, in a subsequent stage with acompound R2—O—Y, where —O—Y is a leaving group and R2 is an organic orinorganic substituent selected such that R2—O—Y is capable of forming,by reaction with the compound (3), the compound according to theinvention of the formula:

In order to be capable of forming the compound of the formula (4) inthis manner, the compound R2—O—Y must in particular not be reactivetowards the halohydrin function:

Moreover, R2—O—Y must react with the terminal phosphate of compound (3)to form the compound (4).

The reaction of the compound of the formula (3) with R2—O—Y is anucleophilic substitution. This reaction is in particular possible andadvantageous for R2 selected from among the group comprising alkyls andalkenes. Y is selected such that R2—O—Y may give rise to the compound(4) by nucleophilic substitution. Y is selected, for example, from amongtosyl, brosyl and triflyl.

A compound according to the invention may accordingly be difunctional ormultifunctional. The phosphohalohydrin function(s) bring(s) about aspecific desired antigenic property towards Tγ9δ2 lymphocytes, and R2 orthe other functional groups of the compound may exhibit other, inparticular therapeutic, properties.

In the case of a compound according to the invention having two or morephosphohalohydrin groups of the formula (1), it is sufficient either tostart from a starting compound having the corresponding number ofphosphorylated alkene groups of the formula (5) and the correspondingchemical structure, or to use the compound of the formula (3) and toreact it with an intermediate compound R2—O—Y having the correspondingnumber of —O—Y functions.

The invention also in particular relates to the novel phosphohalohydrinβ ester compounds of the formula:

where X is a halogen selected from among iodine, bromine and chlorine,

-   -   R1 is selected from among —CH₃ and —CH₂—CH₃,    -   Cat⁺ represents one or more organic or inorganic cation(s)        (including the proton), which may be identical or different in        the same compound,    -   n is an integer between 2 and 20,    -   R3— is selected from among:        -   a halohydrin group of the formula (12),        -   an epoxide group of the formula:

-   -   -   an alkene group of the formula:

-   -   -   m being an integer between 1 and 20.

These compounds (14) are obtained by firstly performing the followinginitial step:

Then, the symmetrical diphosphodihalohydrin compound (14a) is obtainedas follows:

The asymmetrical α,β phosphodiester halohydrin/alkene compound (14b) isobtained as follows:

The asymmetrical α,β phosphodiester halohydrin/epoxide compound (14c) isobtained as follows:

The invention also provides uses of the compounds according to theinvention, in particular the compounds of the formula (2), as primateTγ9δ2 lymphocyte activators, in particular to activate proliferationand/or cytotoxic activity and/or production of mediator substance(s) forprimate Tγ9δ2 lymphocytes with TCR receptors comprising Vγ9 and Vδ2variable regions.

The invention also provides uses of the compounds according to theinvention for the treatment of cells sensitive to primate Tγ9δ2lymphocytes in a natural or artificial medium which may contain Tγ9δ2lymphocytes, in which medium said cells may be brought into contact withTγ9δ2 lymphocytes and which is compatible with the compounds accordingto the invention (i.e. which is not likely to cause the breakdownthereof, at least under certain treatment conditions).

A “cell sensitive to Tγ9δ2 lymphocytes” is taken to mean any cellsubject to the induced effector activity of the Tγ9δ2 lymphocytes: celldeath (cell destruction by Tγ9δ2 lymphocytes); reception of cytokinereleased by Tγ9δ2 lymphocytes (TNF-α, INF-γ . . . ); possibly cellularproliferation induced by Tγ9δ2 lymphocytes.

The invention accordingly provides a Tγ9δ2 lymphocyte activationprocess, in particular a process for activating the proliferation ofTγ9δ2 lymphocytes and/or the cytotoxic activity of Tγ9δ2 lymphocytesand/or the production of mediator substance(s) by Tγ9δ2 lymphocytes, inwhich process these Tγ9δ2 lymphocytes are brought into contact with atleast one compound according to the invention in a medium which containsTγ9δ2 lymphocytes and is compatible with T lymphocyte growth.Advantageously and according to the invention, interleukin, inparticular interleukin-2 (IL-2), is introduced into the medium in aproportion suitable to bring about lymphocyte growth in this medium.Indeed, the presence of the lymphocyte growth factor IL-2 is essentialto achieve proliferation of the T lymphocytes, among which only theTγ9δ2 lymphocytes have been activated by a compound according to theinvention. This growth factor must thus be present in the medium forthose uses in which proliferation of Tγ9δ2 lymphocytes is desired. Thislymphocyte growth factor may pre-exist in the natural state or beinduced or introduced into the medium, simultaneously or not with theincorporation of the compound according to the invention, in the sametherapeutic composition or not. Nevertheless, in certain uses in whichactivation without proliferation of the Tγ9δ2 lymphocytes is desired(for example induced cytotoxicity), the presence of this growth factoris not helpful.

More specifically, the invention provides uses of the compoundsaccording to the invention for therapeutic purposes for the curative orpreventive treatment of pathological conditions producing cellssensitive to primate Tγ9δ2 lymphocytes in a medium which may containthese Tγ9δ2 lymphocytes and in which these cells may be brought intocontact with the Tγ9δ2 lymphocytes.

Advantageously and according to the invention, at least one compoundaccording to the invention is used at a concentration in the mediumwhich brings about activation of polyclonal proliferation of Tγ9δ2lymphocytes. This medium may be selected from among human blood,non-human primate blood, human blood extracts, non-human primate bloodextracts.

Said medium may be extracorporeal, said activation process according tothe invention then being an extracorporeal cellular treatment, inparticular applicable in a laboratory, for studying Tγ9δ2 lymphocytes orthe properties thereof, or for diagnostic purposes. The invention alsoprovides a composition for extracorporeal (ex vivo) diagnostics, whereinit comprises at least one compound according to the invention.

Said medium may also be intracorporeal, activation of the Tγ2δ2lymphocytes then being of therapeutic use.

More particularly, said medium is the peripheral bloodstream of aprimate. The invention accordingly in particular provides a process forthe activation of Tγ9δ2 lymphocytes in the peripheral bloodstream of aprimate, in particular humans, into which is administered at least onecompound according to the invention in a quantity suitable foractivating Tγ9δ2 lymphocytes. At least one compound according to theinvention is thus administered by a general route, in particularparenterally into the peripheral bloodstream.

Said medium may also comprise a cellular site to be treated and at leastone compound according to the invention is administered directly incontact with the cellular site to be treated (topical administration).

The invention accordingly in particular provides therapeutic uses of thecompounds according to the invention for treating pathologicalconditions in primates, said conditions belonging to the groupcomprising cancers, infectious diseases, in particular mycobacterialinfections (leprosy, tuberculosis etc.); parasitic conditions (malariaetc.); pathological immunodeficiency syndromes (AIDS etc.). According tothe invention, a therapeutic composition is administered which issuitable for releasing into the peripheral bloodstream and/or at acellular site to be treated a quantity of at least one compoundaccording to the invention capable of activating Tγ9δ2 lymphocytes.Indeed, it has been demonstrated in general terms in the above-statedprior art that a composition having the property of activating Tγ9δ2lymphocytes may advantageously be used for treating these pathologicalconditions.

As is conventional, throughout the text the terms “therapy” or“therapeutic” encompass not only curative treatment or care, but alsopreventive treatment (prophylaxis), such as vaccination, together withintracorporeal diagnostics (administration for diagnostic purposes).Indeed, by permitting activation of Tγ9δ2 lymphocytes, the inventionallows immunostimulatory treatments which may be advantageous not onlyprophylactically by preventing the development of pathogenic cellssensitive to Tγ9δ2 lymphocytes, but also curatively by inducingdestruction of pathogenic cells sensitive to Tγ9δ2 lymphocytes.

The invention accordingly provides a therapeutic composition comprisingat least one compound according to the invention. More specifically, theinvention relates to a therapeutic composition comprising a quantitycapable of being administered to a primate, in particular in contactwith the peripheral bloodstream or topically, of at least one compoundaccording to the invention, in particular for the preventive or curativetreatment of the above-stated pathological conditions. A compositionaccording to the invention may be an immunostimulant composition or avaccine, the compounds according to the invention being antigens whichactivate Tγ9δ2 lymphocytes.

Advantageously and according to the invention, the therapeuticcomposition is characterized in that it moreover comprises a proportionof interleukin, in particular interleukin-2, suitable for bringing aboutlymphocyte growth in the medium into which it is to be administered.

A therapeutic composition according to the invention may be prepared ina dosage form capable of being administered by a general route, inparticular parenterally directly into the peripheral bloodstream of aprimate, with at least one compound according to the invention in aquantity suitable to activate Tγ9δ2 lymphocytes and one or moreappropriate excipient(s). Given the very low active concentration of thecompounds according to the invention (of the order of 0.1 to 10 nM),such administration may be made without risk of toxicity.

A therapeutic composition according to the invention may also beprepared in a dosage form appropriate for topical administration,directly in contact with the cells sensitive to Tγ9δ2 lymphocytes.

The dosage form of a therapeutic composition according to the inventionis produced in accordance with the selected route of administrationusing conventional pharmaceutical formulation methods. The quantity andconcentration of compound(s) according to the invention and the dosageare determined by reference to known chemotherapeutic methods for thediseases to be treated, taking account of the bioactivity of thecompounds according to the invention towards Tγ9δ2 lymphocytes, theindividual to be treated, the disease in question and the desiredbiological effects.

Advantageously and according to the invention, in the case of abioactive compound at a concentration of between 1 nM and 10 nM, thequantity of compound(s) according to the invention administered by ageneral route is between 0.1 μg and 100 μg, in particular between 1 μgand 10 μg, per kilogram of patient body weight.

It has moreover been demonstrated in vitro that the compounds accordingto the invention exhibit no general toxicity even at concentrations ofup to 100 μM, i.e. of the order of 10⁵ times the bioactiveconcentration. Furthermore, it is known that the biochemical class ofmolecules to which the compounds according to the invention belong(phosphoesters) comprises a family of metabolic compounds found in anyliving cell. The compounds according to the invention thus exhibit notoxic effects other than those induced by the bioactivity thereof uponTγ9δ2 lymphocytes.

Moreover, certain compounds according to the invention have asufficiently low molecular weight (in particular below 500) to becompatible with the elimination thereof via the kidneys and urine.

One example formulation of an injectable therapeutic compositionaccording to the invention for a primate weighing 1 kg is as follows: 5μg of 3-(iodomethyl)-3-butanol-1-yl diphosphate (IHPP) diluted in 0.5 mlof sterile phosphate buffer at pH 7 and adjusted to 37° C.

In this manner, 5 μg of IHPP (compound of the formula (2)) areadministered per 1 kg of animal body weight, corresponding to aconcentration in the circulating blood such as to be greater than thebioactive concentration of IHPP (a concentration of 10 nM of IHPPcorresponding to approx. 5 ng/ml).

It should be noted that the majority of the excipients or otherconventionally used pharmaceutically acceptable additives are chemicallycompatible with the compounds according to the invention.

A therapeutic composition according to the invention may alsoadvantageously comprise one or more other active ingredient(s), inparticular to bring about a synergistic effect. In particular, acompound according to the invention may act as a vaccine adjuvant. Thetherapeutic vaccine composition according to the invention thencomprises a known vaccine composition to which is added a quantity ofcompound(s) according to the invention capable of activating the Tγ9δ2lymphocytes which will not only be able to exert their anti-infectiveactivity directly, but will also be able to activate the T lymphocyteswhich effect the conventional vaccine response.

A therapeutic composition according to the invention may also itselfincorporate primate Tγ9δ2 lymphocytes in a culture in a mediumcompatible with T lymphocyte growth. It may then be used for treatingprimates, or more generally vertebrates with which administration ofprimate Tγ9δ2 lymphocytes may be performed under conditions of immunecompatibility towards said primate Tγ9δ2 lymphocytes. Such a compositionaccording to the invention may be administered by a general route, oreven by a topical route, in contact with target pathogenic cells,sensitive to said primate Tγ9δ2 lymphocytes.

The invention also provides the use of at least one compound accordingto the invention for the production of a therapeutic compositionaccording to the invention. More particularly, the invention relates tothe use of at least one compound according to the invention for theproduction of a therapeutic composition intended for the preventive orcurative treatment of a pathological condition of humans or vertebrateswhich produces cells sensitive to primate Tγ9δ2 lymphocytes, inparticular a pathological condition selected from the group comprisingcancers, infectious diseases, parasitic conditions and pathologicalimmunodeficiency syndromes. To this end, the invention also provides theuse of at least one compound according to the invention for theproduction of a therapeutic composition intended to be administered, inparticular in contact with the peripheral bloodstream or by a topicalroute, to a primate, in particular to humans, for the preventive, orcurative treatment of a pathological condition as mentioned above.

The invention also provides a process for the production of acomposition, in particular a therapeutic composition, according to theinvention having the characteristic of activating Tγ9δ2 lymphocytes, inwhich process at least one compound according to the invention is used.

The invention also relates to a process for the production of atherapeutic composition intended for the preventive or curativetreatment of a pathological condition of humans or vertebrates whichproduces cells sensitive to primate Tγ9δ2 lymphocytes, in which processat least one compound according to the invention is used. The inventionin particular relates to a process for the production of a therapeuticcomposition intended to be administered, in particular in contact withthe peripheral bloodstream or by a topical route, to a primate, for thepreventive or curative treatment of a pathological condition whichproduces cells sensitive to Tγ9δ2 lymphocytes, in particular apathological condition belonging to the group stated above, in whichprocess at least one compound according to the invention is used.

Advantageously and according to the invention, in a production processaccording to the invention, at least one compound according to theinvention is brought into contact with a medium which contains primateTγ9δ2 lymphocytes, and is compatible with T lymphocyte growth, in aquantity suitable to activate these Tγ9δ2 lymphocytes in this medium.Advantageously and according to the invention, said medium comprises asubstance selected from among primate blood and primate blood extracts.A therapeutic composition containing activated Tγ9δ2 lymphocytes is thenobtained so allowing a cellular therapeutic approach to be performed.

It should be noted that the compounds according to the invention arehalogenated and, for this reason alone, cannot correspond to naturalphosphoantigens, in particular to the molecules known as Tubag1, Tubag2,Tubag3 and Tubag4 obtained as described in WO 95/20673. It is in anyevent possible to demonstrate for example that these naturalphosphoantigens are broken down by the bromine water used for thechemical production of the phosphobromohydrins according to theinvention. The compounds according to the invention are thus not naturalantigens, but are synthetic antigens which activate Tγ9δ2 lymphocytes atconcentrations of the same order and with an efficiency similar to oreven greater than that of natural antigens.

It should also be noted that, contrary to the prior art as illustratedby U.S. Pat. No. 5,639,653, which considered that the presence of analkyl or alkene group was essential to activate human Tγ9δ2 lymphocytes,the inventors have observed that by destroying the alkene bond with theaddition of halogen, an element absent from natural biologicalcompounds, the Tγ9δ2 lymphocytes are activated extremely strongly and atvery low concentration. In particular, it may be observed that theeffect may even exceed that of phosphoantigens of natural origin.

Further features, objects and advantages of the invention will beevident from the following Examples, which are provided innon-limitative manner merely for purposes of explanation, and from thefigures:

FIG. 1 is a graph showing the results obtained in Example 10,

FIG. 2 is a graph showing the results obtained in Example 11.

EXAMPLE 1 Production of 3-(bromomethyl)-3-butanol-1-yl diphosphate(BrHPP)

Preparation of 3-methyl-3-butene-1-yl tosylate (isopentenyl tosylate):

2.32 mmol (442 mg) of tosyl chloride and 2.55 mmol (312 mg) of4-(N,N-dimethylamino)pyridine are introduced while stirring with amagnetic stirrer into 5 ml of anhydrous dichloromethane in a glassreaction vessel which is equipped for handling under an inert atmosphereand has been carefully dried. 2.32 mmol (200 mg) of isopentenoldissolved in approx. 1 ml of dichloromethane are slowly added to thismixture through a septum using a syringe. The reaction is monitored bythin-layer chromatography on silica (silica gel 60 F-254; eluent:pentane/ethyl acetate 85/15 vol./vol.; R_(f) (product)=0.4 and R_(f)(TsCl)=0.5). After approx. 3 hours' stirring under a nitrogenatmosphere, the reaction mixture is diluted in a large volume of hexane(approx. 100 ml), resulting in the immediate formation of a whiteprecipitate. The mixture is then filtered and the filtrate concentratedby evaporation under reduced pressure. The solution is then diluted withdiethyl ether and refiltered. Once the solvent has evaporated, ayellowish oil is obtained. The product is purified by preparativechromatography through a silica column (silica gel 60; eluent:pentane/ethyl acetate 85/15). In this manner, 1.98 mmol (475 mg) of3-methyl-3-butene-1-yl tosylate (85% isolated yield) are obtained. Thecompound (colorless oil) is stored at +4° C. in an anhydrous medium.

Preparation of tris(tetra-n-butylammonium) hydrogenpyrophosphate:

4.5 mmol (1 g) of dihydrogenpyrophosphate disodium salt (Na₂H₂P₂O₇) aredissolved in 10 ml of cold deionized water which has previously beenadjusted to pH 9 with a 10 mM ammonia solution. The solution is passedthrough a column containing 19 milliequivalents (4 g) of DOWEX®50-WX8-200 cationic resin (H⁺ form). The acid solution is eluted with15-20 ml of cold deionized water at pH 9. The collected solution isimmediately titrated at pH 7.3 using a 40% aqueous solution oftetra-n-butylammonium hydroxide (Bu₄NOH). After freeze-drying, 4 g oftetra-n-butylanrrnonium salt are obtained as a hygroscopic white solid.The salt is dissolved in 10 ml of anhydrous acetontrile. The solution issubsequently filtered, then dried by evaporation of the solvent underreduced pressure in successive stages. In this manner, a solution oftris(tetra-n-butylammonium) hydrogenpyrophosphate is obtained with apurity of 98% (result deduced from analysis by ionic chromatography(HPAEC)). The volume is adjusted to achieve a salt concentration ofbetween 0.5 and 1 M. The solution is stored at −20° C. in an anhydrousmedium.

Preparation of 3-methyl-3-butene-1-yl diphosphate (isopentenylpyrophosphate):

2.5 ml of a solution of tris(tetra-n-butylammonium)hydrogenpyrophosphate at a concentration of 0.7 M (1.75 mmol) inanhydrous acetonitrile are introduced into a glass reactor which hasbeen dried carefully. The reactor is cooled with an ice bath, then 0.70mmol (168 mg) of 3-methyl-3-butene-1-yl tosylate dissolved in a minimumquantity of acetonitrile (0.5-1 M) are added using a syringe, whilestirring with a magnetic stirrer. Once the tosylate has been introduced,the ice bath is removed, then the reaction is allowed to continue atroom temperature with stirring. The progress of the reaction is thenmonitored by ionic chromatography (HPAEC). After approx. 3 hours, thesolvent is evaporated under reduced pressure and the reaction mediumredissolved in 3 ml of a 98/2 (vol./vol.) water/2-propanol mixture. Thesolution is passed through a column containing 19 milliequivalents (4 g)of DOWEX® 50-WX8-200 cationic resin (NH₄ ⁺ form), then eluted with 10 mlof the water (pH 9)/2-propanol 98/2 (vol./vol.) mixture. Afterfreeze-drying, a white solid containing the crude product is collected.

Purification:

The pyrophosphate and the traces of ammonium monophosphate are separatedfrom the medium by coprecipitation in the presence of ammoniumhydrogencarbonate. The crude product obtained in the preceding stage isdissolved in 4 ml of 0.1 M ammonium hydrogencarbonate, which istransferred into a 25 ml centrifuge tube. The solution is then treatedwith 10 ml of a 1/1 (vol./vol.) acetonitrile/2-propanol mixture byshaling the mixture vigorously (vortex) for a few minutes until a whiteprecipitate has formed. The tube is then centrifuged at 2000 rpm at 10°C. for 5 minutes. The supernatant, into which the organic salts havebeen extracted, is stored at +4° C. The procedure is repeated byredissolving the precipitate in 3 ml of 0.1 M amrnoniumhydrogencarbonate, to which 7 ml of the acetonitrile/2-propanol mixtureare added. The two supernatants are combined and the solvent evaporatedunder a vacuum. An oily liquid is obtained which is stored at +4° C.

The ammonium tosylate is separated from the reaction medium byextraction with the 1/1 (vol./vol.) chloroform/methanol solvent. Theoily liquid from the preceding stage is dissolved in 4 ml of water at pH9 and treated with 1 ml of this solvent by a conventional extractionprocedure which is repeated 3 times. Any traces of solvent are thenremoved from the aqueous phase by evaporation under reduced pressure at30° C. On the basis of analysis by ionic chromatography (HPAEC), an 83%yield of 3-methyl-3-butene-1-yl diphosphate (0.58 mmol, 172 mg) isobtained. The solution is stored at −20° C.

The product is then purified in accordance with requirements by anionexchange chromatography through 360 mg to 10 g Sep-Pak Accell Plus QMA(Waters®) cartridges eluted in succession respectively by 20 mM, 40 mM,100 mM then 200 mM aqueous ammonium hydrogencarbonate solutions, withthe eluted fractions being monitored by chromatography (HPAEC). Thefractions corresponding to the purified product are combined and thenfreeze-dried.

Preparation of 3-(bromomethyl)-3-butanol-1-yl diphosphate:

0.34 mmol (100 mg) of isopentenyl pyrophosphate (ammonium salt)dissolved in 2 ml of neutral pH deionized water are treated under a fumehood at room temperature with 1.9 ml (0.34 mmol) of bromine in asaturated aqueous solution (0.18 M). The bromine solution is addedgradually and preferably to a cold solution of the ammonium salt, withthe mixture being stirred periodically until the bromine water hasbecome colorless. In the event that bromine is added in a slight excess(persistent yellow color), the solution is transferred into a glassflask and then exposed to reduced pressure (rotary evaporator) for a fewminutes at a temperature of 30° C. until the color disappears. Theproduct 3-(bromomethyl)-3-butanol-1-yl diphosphate is producedquantitatively (0.33 mmol, 130 mg); this result being deduced fromanalysis by ionic chromatography (HPAEC). For the purpose of performingbiological testing, the resultant aqueous solutions are filtered andthen neutralized by being passed through a cationic resin column. Thebromide ions may be removed from the solution using a DIONEX® apparatuscomprising an OnGuard®-Ag cartridge attached to an OnGuard®-H cartridge.This apparatus allows selective retention of the halide ions from thesolution. For the purpose of performing biological testing, the aqueoussolutions of the product are sterilized by filtration through a 0.2 μmfilter and stored (preferably with a neutral to slightly acidic pH) at−20° C. In the case of testing performed in vivo, the solutions arepassed beforehand through a DOWEX® 50-WX8-200 cationic resin column (Na⁺form) eluted by two column volumes of deionized water.

EXAMPLE 2 Production of 3-(iodomethyl)-3-butanol-1-yl diphosphate (IHPP)

Preparation of Iodized Water:

A solution of iodized water of the order of 0.5 to 1 mM is prepared byextended sonication (approx. 15 minutes) of a few crystals of iodine ina solution of deionized water and filtration. For tests involving largerquantities, solutions having a higher iodine concentration may beobtained by adding a small proportion of alcohol to the initial aqueoussolution. The iodized water is then titrated with sodium thiosulfateusing starch solution as a color indicator.

Preparation of 3-(iodomethyl)-3-butanol-1-yl diphosphate:

One micromole (1 ml of a millimolar solution) of isopentenylpyrophosphate prepared according to Example 1 in the form of theammonium salt in an aqueous or aqueous/alcohol medium of neutral pH istreated at room temperature by addition of one micromole of iodine in anaqueous solution (1.43 ml of 0.7 mM iodized water). The solution is leftfor 30 minutes at room temperature, then for 30 minutes at +4° C. whileperiodically being stirred vigorously. Once the iodized water has becomecolorless, the product 3-(iodomethyl)-3-butanol-1-yl diphosphate isproduced quantitatively (I micromole in approx. 2.5 ml). The solution isthen treated as in Example 1 for the performance of biological testingand/or for the performance of in vivo testing and stored at −20° C.

EXAMPLE 3 Production of 3-(chloromethyl)-3-butano-1-1-yl diphosphate(C1HPP)

Preparation of Chlorine Water:

The solution of chlorine water is prepared by bubbling gaseous chlorinethrough a solution of deionized water. The solution is then titratedwith sodium thiosulfate in the presence of excess potassium iodide andusing starch solution as a color indicator.

Preparation of 3-(chloromethyl)-3-butanol-1-yl diphosphate:

One micromole (1 ml of a millimolar solution) of isopentenylpyrophosphate prepared according to Example 1 in the form of theammonium salt in an aqueous or aqueous/alcohol medium of neutral pH istreated at room temperature by addition of one micromole of chlorine inan aqueous solution (72 μl of 14 mM chlorine water). After 30 minutes atroom temperature with periodic stirring, the product3-(chloromethyl)-3-butanol-1-yl diphosphate is produced quantitatively(in this case 1 micromole in approx. 1.1 ml). The solution is thentreated as in Example 1 for the performance of biological testing and/orfor the performance of in vivo testing and stored at −20° C.

EXAMPLE 4 Production of 3-(bromomethyl)-3-butanol-1-yl triphosphate(BrHPPP)

Preparation of tetrakis(tetra-n-butylammonium) hydrogentriphosphate:

2.1 mmol (1 g) of tripolyphosphate hexahydrate pentasodium salt(Na₅P₃O₁₀.6H₂O) are dissolved in 10 ml of cold deionized water which haspreviously been adjusted to pH 9 with a 10 mM ammonia solution. Thesolution is passed through a column containing 21 milliequivalents (4.4g) of DOWEX® 50-WX8 cationic resin (H⁺ form). The acid solution iseluted with 20-25 ml of cold deionized water at pH 9. The collectedsolution is immediately titrated at pH 7.0 using a 40% aqueous solutionof tetra-n-butylammonium hydroxide (Bu₄NOH). After freeze-drying, 2.5 gof tetra-n-butylammonium salt are obtained as a hygroscopic white solid.The salt is dissolved in 10 ml of anhydrous acetonitrile. The solutionis subsequently filtered, then dried by evaporation of the solvent underreduced pressure in successive stages. In this manner, a solution oftetrakis(tetra-n-butylamnrnonium) hydrogentriphosphate with a purity of95% is obtained (result deduced from analysis by ionic chromatography(HPAEC)). The volume is adjusted to achieve a salt concentration ofbetween 0.5 and 1 M. The solution is stored at −20° C. in an anhydrousmedium.

Preparation of 3-methyl-3-butene-1-yl triphosphate (isopentenyltriphosphate):

Using the procedure described for the preparation of3-methyl-3-butene-1-yl diphosphate (Example 1), 2 mmol of a molarsolution of tetralcis(tetra-n-butylammonium) hydrogentriphosphate arereacted under a nitrogen atmosphere with 1 mmol (240 mg) of3-methyl-3-butene-1-yl tosylate prepared according to Example 1 in 4 mlof anhydrous acetonitrile for 24 hours. By using aprecipitation/extraction purification procedure similar to that appliedto 3-methyl-3-butene-1-yl diphosphate, on the basis of analysis by ionicchromatography (HPAEC), a yield of 74% of 3-methyl-3-butene-1-yltriphosphate (0.74 mmol, 292 mg) is obtained. Phosphohalohydrincompounds according to the invention are prepared for the purpose ofbiological testing by using a fraction of the product obtained at thisstage which is purified by HPAEC through an IonPac® AS11 column, withtwo or more chromatographic passes being combined. In this manner,approx. 2 ml of an aqueous millimolar solution of neutral pH of3-methyl-3-butene-1-yl triphosphate are prepared in the form of anammonium salt.

Preparation of 3-(bromomethyl)-3-butanol-1-yl triphosphate:

300 nmol (300 μl of a millimolar solution) of isopentenyl triphosphateare treated at room temperature by addition of 300 nmol of bromine in asaturated aqueous solution (1.7 μl of 180 mM bromine water). The colorof the bromine water disappears virtually instantaneously. Once themixture has been stirred and the bromine has become colorless (virtuallyinstantaneously), the product 3-(bromomethyl)-3-butanol-1-yltriphosphate is produced quantitatively (300 μl of a millimolarsolution). The solution is then treated as in Example 1 for theperformance of biological testing and/or for the performance of in vivotesting and stored at −20° C.

EXAMPLE 5 Production of 3-(iodomethyl)-3-butanol-1-yl diphosphate(IHPPP)

300 mmol (300 μl of a millimolar solution) of isopentenyl triphosphateprepared according to Example 4 are treated in an aqueous oraqueous/alcohol medium of neutral pH by addition of 429 μof 0.7 mMiodized water prepared according to Example 2. The solution is left for30 minutes at room temperature while periodically being stirredvigorously. Once the iodized water has become colorless, the product3-(iodomethyl)-3-butanol-1-yl triphosphate is produced quantitatively(729 μl of a 411 μM solution). The solution is then treated as inExample 1 for the performance of biological testing and/or for theperformance of in vivo testing and stored at −20° C.

EXAMPLE 6 Production of α,γ di-[3-(bromomethyl)-3-butanol-1-yl]triphosphate (diBrHTP)

Preparation of α,γ di-[3-methyl-3-butene-1-yl] triphosphate:

Using the procedure described for the preparation of3-methyl-3-butene-1-yl diphosphate (Example 1), 0.5 mmol of a molarsolution of tetrakis(tetra-n-butylammonium) hydrogentriphosphate(prepared according to Example 4) are reacted under a nitrogenatmosphere with 1 mmol (240 mg) of 3-methyl-3-butene-1-yl tosylate(prepared according to Example 1) in 4 ml of anhlydrous acetonitrile for24 hours. By using a precipitation/extraction purification proceduresimilar to that applied to 3-methyl-3-butene-1-yl diphosphate, on thebasis of analysis by ionic chromatography (HPAEC), a yield of 81% of α,γdi-[3-methyl-3-butene-1-yl] triphosphate (0.4 mmol, 178 mg) is obtained.Phosphohalohydrin compounds according to the invention are prepared forthe purpose of biological testing by using a fraction of the productobtained at this stage which is purified by HPAEC through an Ionpac®AS11 column, with two or more chromatographic passes being combined.Before each chromatographic pass and in order to improve isolation ofthe product, the fraction to be purified is treated enzymatically withalkaline phosphatase in order to break down the isopentenyl triphosphatewhich is a secondary product of the reaction. In this manner, approx. 1ml of an aqueous millimolar solution of neutral pH of α,γdi-[3-methyl-3-butene-1-yl] triphosphate are prepared in the form of anammonium salt.

Preparation of α,γ di-[3-(bromomethyl)-3-butanol-1-yl] triphosphate:

250 nmol (250 μl of a millimolar solution) of α,γdi-[3-methyl-3-butene-1-yl] triphosphate are treated at room temperatureby addition of 250 nmol of bromine in a saturated aqueous solution (1.4μl of 180 mM bromine water). Once the mixture has been stirred and thebromine has become colorless (virtually instantaneously), the productα,γ di-[3-(bromomethyl)-3-butanol-1-yl] triphosphate is producedquantitatively (approx. 250 μl of a millimolar solution). The solutionis then treated as in Example 1 for the performance of biologicaltesting and/or for the performance of in vivo testing and stored at −20°C.

EXAMPLE 7 Production of α,γ di-[3-(iodomethyl)-3-butanol-1-yl]triphosphate (diIHTP)

250 nmol (250 μl of a millimolar solution) of α,γdi-[3-methyl-3-butene-1-yl] triphosphate prepared according to Example 6are treated in an aqueous or aqueous/alcohol medium of neutral pH byaddition of 358 μl of 0.7 mM iodized water prepared according to Example2. The solution is left for 30 minutes at room temperature whileperiodically being stirred vigorously. Once the iodized water has becomecolorless, the product α,γ di-[3-(iodomethyl)-3-butanol-1-yl]triphosphate is produced quantitatively (608 μl of a 411 μM solution).The solution is then treated as in Example 1 for the performance ofbiological testing and/or for the performance of in vivo testing andstored at −20° C.

EXAMPLE 8 Production of Uridine 5′-triphosphateγ-[3-(iodomethyl)-3-butanol-1-yl]

Preparation of Uridine 5′-triphosphate y-[3-methyl-3-butene-1-yl]:

This product is prepared in accordance with the procedure described byKNORRE D. C. et al. “General Method for the synthesis of ATPGamma-derivatives” Febs Letters, 1976, 70-1, 105-108, starting from 40μmol of uridine 5′-triphosphate (UTP) (triethylammonium salt) in thepresence of an excess of isopentenol. Phosphohalohydrin compoundsaccording to the invention are prepared for the purpose of biologicaltesting by purifying a fraction of the product obtained by HPAEC throughan IonPac®) AS11 column, with two or more chromatographic passes beingcombined. Before each chromatographic pass and in order to improveisolation of the product, the fraction to be purified is treatedenzymatically with alkaline phosphatase in order to break down thesecondary products (UDP and UMP) and unreacted UTP. In this manner,approx. 500 μl of an aqueous 300 μM solution of neutral pH of uridine5′-triphosphate γ-[3-methyl-3-butene-1-yl] are prepared in the form ofan ammonium salt.

Preparation of Uridine 5′-triphosphateγ-[3-(iodomethyl)-3-butanol-1-yl]:

75 nmol (250 μl of a 300 μM solution) of uridine 5′-triphosphateγ-[3-methyl-3-butene-1-yl] in ammonium salt form are treated in anaqueous medium of neutral pH by addition of 108 μl of 0.7 mM iodizedwater prepared according to Example 2. The solution is left for 20minutes at room temperature while periodically being stirred vigorously.Once the iodized water has become colorless, the product uridine5′-triphosphate γ-[3-(iodomethyl)-3-butanol-1-yl] is producedquantitatively (approx. 360 μl of a 200 μM solution). The solution isthen treated as in Example 1 for the performance of biological testingand/or for the performance of in vivo testing and stored at −20° C.

EXAMPLE 9 Production of α,β, di[3-bromomethyl-3-butanol-1-yl]diphosphate

This product is prepared in accordance with a procedure analogous tothat described in Example 6, with tetrakis(tetra-n-butylammonium)hydrogentriphosphate being replaced by tris(tetra-n-butylammonium)hydrogenpyrophosphate (prepared according to Example 1) for thepreparation of the intermediate compound: α,βdi-[3-methyl-3-butene-1-yl] diphosphate.

EXAMPLE 10 Measurement of Antigenic Activity by Stimulation of theProliferation of Tγ9δ2 lymphocytes in a Culture

20 microliters of the aqueous solution of the compound according to theinvention adjusted to the final concentration specified in the test areadded to an in vitro culture of 10⁶ total T lymphocytes in 1 ml,separated from the blood of a healthy adult human donor and initiallycontaining 1-5% of Tγ9δ2 lymphocytes, in an adequate culture medium(RPMI 1640+10% of inactivated human serum and 50 U/ml of humaninterleukin-2 (hIL-2)). After culturing for 4 days, 50 U of dIL-2 areadded per milliliter of culture medium. After 8 days, the cells arecounted, collected, washed with phosphate buffer, and the Tγ9δ2 typecells are detected in the culture by labeling with conventionalcommercial reagents (fluorescein-labeled monoclonal antibodies) and theproportion thereof determined by flux cytometry analysis. The parametermeasured is either the change in the proportion of or the increase inthe number of Tγ9δ2 cells in cultures in the presence of the compoundaccording to the invention in comparison with cultures not containing acompound according to the invention. The results of this testing arerepresented by plotting curves of these values (y-axis in FIG. 1) as afunction of concentration on a logarithmic scale of the compoundaccording to the invention placed in the culture (x-axis in FIG. 1).

FIG. 1 shows the results obtained with the compounds according to theinvention obtained in Examples 1 (BrHPP) and 2 (IHPP), with the dottedline being a negative control (value obtained in the absence of thecompound according to the invention).

Table 1 below shows the ED50 values, the effective dose at 50% of themaximum polyclonal lymphocyte amplification effect obtained as statedabove with various compounds according to the invention.

TABLE I MOLECULE ED 50% Name Abbreviation Structure nM isopentenylpyrophosphate IPP

3000 3-(chloromethyl)- 3-butanol-1-yl diphosphate CIHPP

100 3-(bromomethyl)- 3-butanol-1-yl diphosphate BrHPP

10 3-(iodomethyl)-3- butanol-1-yl diphosphate IHPP

3 3-(bromomethyl)- 3-butanol-1-yl triphosphate BrHPPP

120 3-(iodomethyl)-3- butanol-1-yl triphosphate IHPPP

70 α, γ di-3- (bromomethyl)-3- butanol-1-yl triphosphate di-BrHTP

3000 α, γ di-3- (iodomethyl)-3- butanol-1-yl triphosphate di-IHTP

7000

EXAMPLE 11 Measurement of Antigenic Activity by Stimulation of InducedCytotoxicity

The specific cytotoxic activity of a Tγ9δ2 lymphocyte clone measured inaccordance with the induced cytotoxicity test is compared, said activitybeing stimulated with decreasing concentrations of the phosphoantigenTubag3 obtained as described by WO 95/20673 (curve represented by blackdiamonds in FIG. 2), of the compound BrHPP according to the inventionobtained in Example 1 (curve represented by black squares in FIG. 2), ofthe compound IHPP according to the invention obtained in Example 2(curve represented by white circles in FIG. 2) and of isopentenylpyrophosphate IPP (curve represented by black triangles in FIG. 2).

It may be noted that the compounds according to the invention are activeat a concentration of the order of 10 nM, which is of the same order asthat of the natural phosphoantigen Tubag3, whereas the prior artcompound IPP is active at a concentration of the order of 3 μM, or 300times higher.

EXAMPLE 12 Toxicity of BrHPP (Sodium Salt)

Eight 30 g mice received an intravenous injection (caudal vein) of 300μl of PBS buffer containing 1 mg of BrHPP (sodium salt). No sign ofshock or pyrogenicity is observed: 8 mice are still alive after 30 days;no significant variation in the weight of the mice is recorded duringthe study. Toxicity is thus less than 12.5% for a dose of 33.4 mg ofBrHPP (Na⁺) per kilogram of animal body weight.

EXAMPLE 13 Pyrogenicity of BrHPP (Sodium Salt)

This test is performed in accordance with a protocol specified bystandards ISO-10993-11 (1993) and USP XXIII. The test involves assessingthe febrile reaction in rabbits after intravenous administration ofBrHPP (sodium salt) at 400 μg/ml (i.e. 10 ml of extract per kilogram) tothree rabbits. Temperature is taken every 30 minutes for 3 hoursstarting 30 minutes after the injection.

Only one rabbit exhibited an increase in temperature of greater than0.5° C. (minimum threshold). On injection of the 400 μg/ml solution, therabbits exhibited a side-effect talding the form of muscle tremors.Another rabbit injected with the same solution diluted 1:40, namely to10 μg/ml, in 19 minutes exhibited no reaction to the injection.

EXAMPLE 14 In vivo Bioactivity of BrHPP (Sodium Salt) in the Macaque

These tests are performed on monkeys of the species Macaca fascicularis(males weight 3-4 kg) anaesthetized with 15 mg/kg Zoletil(zolazepam)®/0.01 mg/kg atropine. The batches of BrHPP (in NA⁺ form) aresubjected to prior purity testing (>99%), quantified and have beentested in accordance with the pyrogenicity tests of Example 12. Theanimals are injected by rapid intravenous injection (2-5 minutes intothe saphenous vein) with 0.1 mg/kg of BrHPP (i.e. 0.3-0.4 mg per animal)diluted in 10 ml of Ringer lactate. Each animal also receives asubcutaneous injection of 90,000 units of interleukin-2 (IL-2). Theseinjections are performed once daily for four days. Control animalsreceive only the subcutaneous injection of IL-2 once daily for fourdays.

The proportion of Tγ9δ2 lymphocytes in total lymphocytes in the variousanimals is compared in the following three situations:

-   -   in the animal's bloodstream,    -   in a culture of total lymphocytes taken from the animal        receiving only BrHPP,    -   in a culture of total lymphocytes talken from the animal        receiving BrHPP+IL-2.        The results are shown in Table II below:

TABLE II Reactivity ex vivo of Tγ9δ2 % of Tγ9δ2 lymphocytes lymphocytes(% after 10 days' culturing in in the blood vitro) in vivo AmplificationAmplification Animals (7 days after by 40 nM by 40 nM injected:injection) BrHPP BrHPP + IL-2 Controls or ~1%* [0.8-1.4] 1% ~8% [4.7-12]IL-2 only BrHPP only ~1% 1% ~11% [10-12] BrHPP only, ~1% 1% 40.2% twice(1 injection + 1 booster) BrHPP + IL-2 7% [5-10] 10% 50.0%

It may be noted that the single or repeated intravenous injection ofBrHPP alone preactivates the monkey's Tγ9δ2 lymphocytes, but does notstimulate the growth thereof in the animal. The injection of BrFPP alonebrings about preactivation of the Tγ9δ2 lymphocytes, which is inparticular manifested by expression of the growth factor IL-2 from thesurface of the receptor cells. In contrast, intravenous injection ofBrHPP in the presence of subcutaneous IL-2 preactivates these same cellsand brings about the growth thereof in vivo. Injection of the growthfactor IL-2 alone brings about no specific response in the Tγ9δ2lymphocytes.

1. A compound selected from the group consisting of:

wherein: X is a halogen; n is 2 to 20; Cat⁺ is a cation; R1 is methyl orethyl; and

where X is a halogen selected from the group consisting of iodo, bromo,and chloro substituent, R1 is methyl or ethyl, Cat⁺ is a cation, n is aninteger between 2 and 20 R3 is: a halohydrin group of the formula:

an epoxide group of the formula:

an alkene group of the formula:

m being an integer between 1 and
 20. 2. The compound according to claim1, wherein n is 2 and R1 is methyl.
 3. The compound according to claim1, wherein said compound is


4. The compound according to claim 3, wherein n is 2 and R1 is methyl.5. The compound according to claim 1, wherein said compound is


6. The compound of claim 5, wherein n is 2 and R1 is methyl.
 7. Acomposition comprising an excipient and a compound according to claim 1.8. The composition according to claim 7, wherein said compound is


9. The composition according to claim 8, wherein n is 2 and R1 ismethyl.
 10. The composition according to claim 8, wherein n is 2, R1 ismethyl and X is Br.
 11. The composition according to claim 7, whereinsaid compound is


12. The composition according to claim 11, wherein n is 2 and R1 ismethyl.
 13. A composition comprising from 0.1 μg to 100 μg of a compoundaccording to claim 1 and an excipient.
 14. The composition according toclaim 13, wherein said compound is


15. The composition according to claim 14, wherein n is 2 and R1 ismethyl.
 16. The composition according to claim 14, wherein n is 2, R1 ismethyl and X is Br.
 17. The composition according to claim 13, whereinsaid compound is


18. The composition according to claim 17, wherein n is 2 and R1 ismethyl.
 19. A method of inducing an immune response to a vaccine antigencomprising the administration of a composition comprising a vaccinecomposition and a compound according to claim
 1. 20. The methodaccording to claim 19, wherein said compound is


21. The method according to claim 20, wherein is 2, R1 is methyl and Xis Br.
 22. The method according to claim 20, wherein n is 2 and R1 ismethyl. R1 is methyl and X is Br.
 23. The method according to claim 19,wherein said compound is


24. The method according to claim 23, wherein n is 2 and R1 is methyl.25. A method of activating Tγ₉δ₂ lymphocytes in vivo, comprisingadministering to a patient in need thereof a composition comprising aneffective amount of IL-2 in combination with an effective amount of aphosphohalohydrin compound according to claim
 1. 26. The methodaccording to claim 25, wherein said compound is


27. The method according to claim 26, wherein n is 2 and R1 is methyl.28. The method according to claim 26, wherein n is 2, R1 is methyl and Xis Br.
 29. The method according to claim 25, wherein said compound is


30. The method according to claim 29, wherein n is 2 and R1 is methyl.31. The method according to claim 25, wherein said compound is


32. The method according to claim 31, wherein n is 2 and R1 is methyl.33. The method according to claim 31, wherein n is 2, R1 is methyl and Xis Br.